Multidrug multiligand conjugates for targeted drug delivery

ABSTRACT

Described is a multi drug multiligand conjugate for targeted drug delivery. The MDML conjugate contains a plurality of tripartite molecules linked to a central scaffold moiety, with each tripartite molecule comprising a targeting molecule, a therapeutic agent and a scaffold binding element. The MDML conjugate allows for more efficient delivery of therapeutic agents to the cells resulting in enhanced therapeutic efficiency. A model MDL conjugate is disclosed as well as method for the synthesis of the model conjugate.

This application is a divisional of U.S. patent application Ser. No.10/281,840, filed Oct. 28, 2002, which claims the benefit of U.S.provisional application No. 60/348,229, filed Oct. 26, 2001. The presentdisclosure relates generally to the field of drug delivery.

BACKGROUND OF THE INVENTION

1. Field of the Invention

2. Description of the Related Art

Targeted drug delivery is a powerful strategy in the treatment anddiagnosis of various human diseases, such as cancer, bacterial and viralinfections and any disease expressing specific and targetable receptors,antigens or other markers. Targeted drug delivery has gained momentumduring the past several years based on the specific delivery therapeuticmolecules to malignant tissues comprising the disease state, such as thedelivery of oncolytic agent to the neoplastic tissue. The ability tospecifically target therapeutic molecules to specific cells of interesthas advanced considerably during the past several years with thedevelopment of monoclonal antibodies and of synthetic small moleculepeptides (SSMPs) capable of binding to tumor cell surface receptors. Anumber of drug, toxin, and radioisotope conjugates of tumor-recognizingmolecules have been developed with some currently in clinical use.

Allergic reactions against large-molecule antibodies in humans due tothe immunoreactivity of these proteins have hampered the desireddevelopment of antibody-drug conjugates for targeted delivery and muchattention has been focused on the SSMPs which usually show noimmunoreactivity due to smaller molecular sizes. With the exception ofradiolabeled peptides, SSMPs have not been fully utilized as targetingdrug delivery systems. Recently, the use of SSMPs in a targeted drugdelivery systems with a paclitaxel-bombesin conjugate as a modelcompound was demonstrated (Safavy, U.S. Pat. No. 6,191,290, 2001; Safavyet al., J Med Chem, 1999, 42, 4919-4924). This and similar moleculeswill be referred to as single drug, single ligand (SDSL) conjugates.While effective at specifically delivering the therapeutic molecule tothe target cell of interest, these SDSL conjugates have severallimitations. First, the biological half-life of these SDSL conjugates isquite short, with the conjugates undergoing rapid clearance from thecirculation (usually on the order of 1 to 5 minutes). As a result, onlya fraction of the SDSL conjugates are taken up by the target cells,which results in a low therapeutic efficiency. Secondly, the ability toadapt SDSL conjugates to various disease states or to deliver differenttherapeutic molecules is limited. It would be beneficial if a conjugatewere provided that is designed to deliver different therapeuticmolecules to the target cells with a minimum of experimentalmanipulation.

Therefore, it would be advantageous to provide a pharmaceutical compoundfor use in a single-step drug delivery method that is capable ofspecifically delivering to target cells (via a targeting conjugate)multiple copies of the same or different therapeutic agent(s). Thisapproach may result in higher therapeutic indices which may bring abouta more significant response from the target cells. The presentdisclosure provides such a strategy and provides a method for thesynthesis of a model conjugate as proof-of principle. This modelconjugate was used in a tumor cell proliferation assay to demonstratethe drug-enhancing potential of this approach.

SUMMARY

The present disclosure shows the synthesis and design of two embodimentsof a multidrug multiligand (MDML) conjugate which is capable ofspecifically delivering multiple doses of a therapeutic molecule to atarget cell.

In one embodiment of the present disclosure, there is provided apharmaceutical compound comprising a plurality of tripartite moleculesin combination with a scaffold moiety. The tripartite moleculescomprises a targeting molecule, a therapeutic agent, a scaffold bindingelement linked together by a central core molecule, the tripartitemolecule being capable of binding to the scaffold moiety throughcomplementary binding between the scaffold binding element and thescaffold moiety.

In another embodiment of the present disclosure, alternate methods ofsynthesizing the MDML conjugates are described. Specifically, thesynthesis of one embodiment of the MDML conjugates, incorporatingpaclitaxel (a taxane derivative) as the therapeutic agent, abombesin-derived peptide, BBN [7-13] as the targeting molecule, lysineor diamino benzoic acid as the core molecule and streptavidin/biotin asthe scaffold moiety/scaffold binding element (the PTX-BBN[7-13]-BTN/SaVconjugate), is described in detail.

In still yet another embodiment of the present disclosure there isprovided a method of treating an individual having a disease byadministering the MDML conjugates of the present disclosure to anindividual in an amount sufficient to treat the disease. Because of thehigher therapeutic index achieved by the MDML conjugates, they may beadministered at a lower concentration range than that of theun-conjugated therapeutic agents by means of injection or intravenousinfusion. By disease it is meant any abnormal condition of an organ orother part of an organism.

In another embodiment of the present disclosure, there is provided amethod of administering the MDML conjugates of the present disclosure toan individual as a diagnostic reagent. Because of the higher therapeuticindex achieved by the MDML conjugates, they may be administered at alower concentration range than that of the un-conjugated therapeuticagents by means of injection or intravenous infusion.

In still another embodiment of the present disclosure, a kit is providedto aid in the treatment of individuals having a particular disease

Other and further aspects, features, and advantages of the presentdisclosure will be apparent from the following description of thepresently preferred embodiments of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

So that the features, advantages and objects of the disclosure willbecome clear, are attained and can be understood in detail, reference ismade to the appended drawings, which are described briefly below. It isto be noted, however, that the appended drawings illustrate preferredembodiments of the disclosure and therefore are not to be consideredlimiting in their scope.

FIG. 1 shows the synthesis of two embodiments of the PTX-BBN[7-13]-BTN/SaV conjugate, one embodiment of the MDML conjugate of thepresent disclosure, utilizing paclitaxel (taxol) as the therapeuticagent, a bombesin derivative, BBN [7-13] as the targeting molecule andstreptavidin/biotin as the scaffold moiety/scaffold binding element.FIG. 1A shows the synthesis of PTX-BBN[7-13]-BTN/SaV using lysine as thecore molecule and FIG. 1B shows the synthesis of PTX-BBN[7-13]-BTN/SaVusing diamino benzoic acid as the core molecule;

FIG. 2 shows an increase in molecular weight of thePTX-BBN[7-13]-BTN/SaV MDML conjugate described in FIG. 1A. Aliquots ofthe synthetic reaction described in FIG. 1A were taken (A, sample ofstreptavidin added at step h; B, aliquot of compound 9; C, aliquot ofcompound 10) and analyzed by SE-HPLC;

FIG. 3 shows a general schematic representation of the MDML conjugatesof the present disclosure;

FIG. 4 shows a schematic model of the mechanism of action of thePTX-BBN[7-13]-BTN/SaV MDML conjugate described;

FIG. 5 shows a cell proliferation assay of NCI-HI229 non-small cell lungcancer cells at 4 days post treatment with 50 nM each of the followingfor three hours: 1) untreated cells (Untreated); 2) unconjugatedpaclitaxel (PTX); 3) biotin-streptavidin complex (BTN/SaV); 4) a mixtureof BTN/SaV and PTX; and 5) the PTX-BBN[7-13]-BTN/SaV conjugate(Conjugate) described in FIG. 1A. The PTX-BBN[7-13]-BTN/SaV conjugatedemonstrated 37% growth inhibition compared to 0% growth inhibition byfree PTX and untreated cells, and 1% and 4% inhibition by BTN/SaV andBTN/SaV+PTX controls, respectively; and

FIG. 6 shows an apoptosis (programmed cell death) induction assay ofNCI-HI229 non-small cell lung cancer cells at 4 days post treatment with50 nM each of the following for three hours: 1) untreated cells(Untreated); 2) unconjugated paclitaxel (PTX); 3) biotin-streptavidincomplex (BTN/SaV); 4) a mixture of BTN/SaV and PTX; and 5) thePTX-BBN[7-13]-BTN/SaV conjugate (Conjugate) described in FIG. 1A.Apoptosis was measured by an Annexin V-FITC kit. ThePTX-BBN[7-13]-BTN/SaV conjugate showed a 7% induction of apoptosis, ascompared to about 2.0%. 2.3%, 2.5% and 2.4% induction for untreatedcells, free PTX, the BTN/SaV complex and the BTN/SaV+PTX controls,respectively.

DETAILED DESCRIPTION

The present disclosure illustrates that the effect of a giventherapeutic agent may be enhanced by incorporation of the therapeuticagent into a MDML conjugate capable of delivering a plurality oftherapeutic agents to specific target cells of interest by virtue of aplurality of targeting molecules incorporated into the same conjugate.The present disclosure describes methods for the design, synthesis anddelivery of MDML conjugates for the treatment of various human diseasestates. Cancer is the exemplary human disease discussed below and citedin the examples, but this disclosure should not be interpreted to limitonly to the treatment of cancer as one of ordinary skill in the artwould be able to adapt the teachings of the disclosure to treatment ofalternate disease states. It will be understood by those of skill in theart that the MDML conjugate described can be used to treat a variety ofdisease states by incorporating the desired targeting molecule(s) andtherapeutic agent(s).

The MDML conjugate described in the present disclosure comprise aplurality of tripartite molecules in combination with a scaffold moiety.The tripartite molecules each comprise a targeting molecule, atherapeutic agent, a scaffold binding element linked together by acentral core molecule. The core molecule provides the framework forconstruction of the tripartite molecule. The core molecule can be anymolecule that provides sufficient functional groups to link together thetargeting molecule, the therapeutic agent and the scaffold bindingelement in a functional manner. The functional groups should bepositioned in a manner to minimize steric hindrance between thetargeting molecule, the therapeutic agent and the scaffold bindingelement. In one embodiment, the core molecule is the amino acid lysine.In an alternate embodiment, the core molecule is diamino benzoic acid.Other core molecules may be used as described.

The tripartite molecule is capable of binding to the scaffold moietythrough complementary binding between the scaffold binding element andthe scaffold moiety. The scaffold moiety provides the framework to jointhe tripartite molecules together and allows for the delivery ofmultiple doses of the therapeutic agent to the target cell of interest.

The targeting molecule is any molecule that can direct the MDMLconjugate to a defined population of cells of interest (target cells).The targeting molecule may be, but is not limited to, a peptide, areceptor ligand peptide (meaning a peptide that is designed tospecifically bind to one or more designated receptor molecules), anantibody or antibody fragment. Exemplary targeting molecules include,but are not limited to, bombesin/gastrin-releasing peptide (BBN/GRP)receptor-recognizing peptide, a somatostatin receptor recognizingpeptide, an epidermal growth factor receptor recognizing peptide, amonoclonal antibody or a portion thereof, or polyclonal antibody or aportion thereof, or a receptor recognizing carbohydrate, or anycombination of the above. Examples of receptor recognizing polypeptidesthat bind to the BBN/GRP receptor and/or the somatostatin receptor arethe BBN[7-13] (SEQ ID NO. 1) peptide, the BNN[7-14] (SEQ ID NO. 2)peptide and a semi-synthetic derivative of BBN as shown in SEQ ID NO. 3.The targeting molecule incorporated into each tripartite molecule may bethe same or may be different. The targeting molecule need not direct theMDML conjugate to only one cell type. The selection of a particulartargeting molecule will depend on the cell population to be targeted andis within the ordinary skill in the art.

The therapeutic agent can be any diagnostic agent or any pharmaceuticalagent, or a combination thereof. Therapeutic agents include, but notlimited to, drugs, anti-tumor agents, toxins, radionuclides, metallicnuclei or a combination of the above. Exemplary therapeutic agentsinclude taxane molecule. By taxane molecule it is meant to include anytaxane derivatives such as paclitaxel (taxol) and toxotere (docetaxel)and their analogues. In one embodiment describe, the therapeutic agentis paclitaxel. The therapeutic agent incorporated into each tripartitemolecule may be the same or may be different. The selection of aparticular therapeutic agent will depend on the particular disease to betreated or condition to be diagnosed and is within the ordinary skill inthe art.

The linker molecules may be any molecule capable of linking thetargeting molecule, the therapeutic agent and the scaffold bindingmoiety to a central molecule. The linker molecules may reduce theeffects of steric hindrance and increase the overall yield of thesynthetic reaction. However, the use of a linker molecule is optional.In the embodiment described polyethylene glycol (PEG) was used as thelinker. The chemical and biological properties of PEG molecules havebeen extensively studied and some useful characteristics of this polymerhave been noted. These include aqueous as well as organic solubilities,lack of immunogenicity, and favorable blood clearance patterns and invivo behavior. Furthermore, PEG molecules are available in a wide rangeof chemistries, facilitating the formation of the tripartite molecules.In the embodiment disclosed, a heterobifunctional derivative of the PEGwas used in this synthesis, allowing the PEG to connect the lysine andtargeting molecule, therapeutic agent and scaffold binding moiety asdescribed below in Example 1 and FIG. 1A.

The scaffold binding element and the scaffold moiety may be any set ofmolecules that are capable of specific, complementary, covalent ornon-covalent binding to one another. Exemplary binding pairs include,but are not limited to, poly(amino acids), peptides, polypeptides,proteins, carbohydrates, biotin/avidin, biotin/streptavidin,hapten/antibody, ligand/receptor, enzyme/inhibitor andlectin/carbohydrate. The biotin/streptavidin system has been used inthis disclosure as the model scaffold binding element/scaffold moieties.Avidin (isolated from egg whites) and streptavidin (isolated fromStreptomyces avidinii), each have a very high binding affinity forbiotin. The streptavidin-biotin system represents the strongestnon-covalent biological interaction known between a protein and aligand. Streptavidin has a lower non-specific tissue binding andabsorption. Both avidin and streptavidin are capable of binding 4molecules of biotin. Modified forms of avidin and streptavidin may alsobe used in methods of this disclosure. Biotin includes, but is notlimited to biotin which has been modified by the addition of alkylgroups, and biotin derivatives such as active esters, amines, hydrazidesand thiol groups with the complimentary reactive groups on polymersbeing amines, acyl and alkyl leaving groups, carbonyl groups and alkylhalides or Michael-type acceptors.

FIG. 3 shows the generic schematic representation of the MDML conjugatesof the present disclosure. The generic MDML conjugates comprise ascaffold moiety to which a plurality of therapeutic agent (Tax, with xbeing the number of therapeutic molecules attached) and targetingmolecules (TMy, with y being the number of targeting molecules attached)are attached via a scaffold binding moiety. The targeting molecules andtherapeutic agents can be attached directly to the scaffold moiety, orcan be attached to the scaffold moiety through a linker element. Due tosteric effects encountered during synthesis, it is preferred that alinker element be employed (shown as a straight line in FIG. 3). Theplurality of targeting molecules allow the MDML conjugate to belocalized to specific target cells, whereby the plurality of therapeuticagents are delivered to the cell. In this manner, increasedconcentrations of the therapeutic agents are delivered to the cell,which may bring about a more significant response of the cells to thetherapeutic agents. The therapeutic agents attached to the scaffoldmoiety may be the same or different. The selection of therapeutic agentto be used, and the combination of therapeutic agents to be used ifdesired, will depend upon the target cell selected. Such selection iswithin the ordinary skill in the art of those in the field. In the samemanner, the selection of the specific targeting molecule(s) to be usedwill also be determined by the target cell selected. Again, selection ofthe appropriate targeting molecule is within the ordinary skill in theart of those in the field.

FIG. 4 illustrates a schematic model of tumor uptake of the model MDMLconjugate PTX-BBN[7-13]-BTN/SaV. Individual tripartite molecules aresynthesized according to the scheme described in FIGS. 1A and 1B andExample 1 below. The tripartite molecules comprise a core molecule towhich is covalently linked the bombesin-derived peptide BBN[7-13] whichserves as the targeting molecule, paclitaxel, (PTX) as the therapeuticagent and biotin (BTN) as the scaffold binding element. The BBN[7-13],paclitaxel and biotin are linked to the core molecule by PEG linkerelements. Once synthesized, the tripartite molecules are joinednon-covalently to a streptavidin (SaV) which serves as the scaffoldmoiety, to form the MDML conjugate PTX-BBN[7-13]-BTN/SaV. As discussedabove, the streptavidin scaffold can bind 4 tripartite molecules. ThePTX-BBN[7-13]-BTN/SaV conjugate contains multiple copies of the same ordifferent therapeutic agents. In addition, the PTX-BBN[7-13]-BTN-SaVconjugate may contain the same or different targeting molecules. In oneembodiment, the same targeting molecules are employed. In an alternateembodiment, different targeting molecules are used, with the differenttargeting molecules being directed to the same or different targetcells. The PTX-BBN[7-13]-BTN-SaV conjugate circulates through the bodyuntil BBN[7-13] targeting molecules detect and bind to their bindingtarget on the tumor cell, in this case the bombesin receptor. Once boundto the tumor cell, the therapeutic agents act on the tumor cell throughinternalization or proximity to the cell, causing cell damage andultimately cell death.

As can be seen in FIGS. 5 and 6, the PTX-BBN[7-13]-BTN-SaV conjugate isactive against tumor cells in vitro. FIG. 5 shows a cell proliferationassay demonstrating the sensitivity of native, untransfected NCI-H1299non-small cell lung cancer cells to the PTX-BBN[7-13]-BTN-SaV conjugate.Cells were grown as described in Example 2 and exposed to varioustreatments. The cells were left untreated or treated with 50 nM of thePTX-BBN[7-13]-BTN-SaV conjugate, unconjugated paclitaxel (PTX), thebiotin/streptavidin complex (BTN/SaV) or a mixture of BTN/SaV and PTX.After 3 hours, the media containing these additives was removed andreplaced with fresh media. After 96 hours, the cells were removed fromthe 24 well plates by treatment with trypsin/EDTA and counted with aCoulter particle counter. All treatments were done in quadruplicate andthe cell counts were normalized to the untreated cells. As can be seenin FIG. 5, the PTX-BBN[7-13]-BTN/SaV conjugate inhibited cell growth by37%, as compared to 0% growth inhibition by free PTX, and 1% and 4%inhibition by BTN/SaV and BTN/SaV+PTX controls, respectively.

FIG. 6 illustrates the results of an apoptosis induction assay,illustrating NCI-HI299 cells are induced to undergo apoptosisspecifically in response to the PTX-BBN[7-13]-BTN-SaV conjugate. Cellswere grown as described above and in Example 3 and left untreated ortreated with 50 nM of the PTX-BBN[7-13]-BTN-SaV conjugate, unconjugatedpaclitaxel (PTX), the biotin/streptavidin complex (BTN/SaV) or a mixtureof BTN/SaV and PTX. After 3 hours, the media containing these additiveswas removed and replaced with fresh media. After 96 hours, the cellswere removed from the 24 well plates by treatment with trypsin/EDTA andanalyzed for Annexin V by flow cytometry using an Annexin V-FITC kit(Biovision, Palo Alto, Calif.). All treatments were done in triplicateand presented as the percentage of cells staining with Annexin V-FITC.The PTX-BBN[7-13]-BTN/SaV conjugate showed a 7% induction of apoptosis,as compared to about 2.0%. 2.3%, 2.5% and 2.4% induction for untreatedcells, free PTX, the BTN/SaV complex and the BTN/SaV complex+PTXcontrols, respectively.

The experiments shown in FIGS. 5 and 6 and described in Examples 2 and 3demonstrate the therapeutic effect of the PTX-BBN[7-13]-BTN/SaVconjugate, and show that the MDML conjugates provide an improvedapproach to drug delivery and treatment of various disease states. Ineach of the experiments, the free paclitaxel has no or negligible effecton cell growth or induction of apoptosis, while thePTX-BBN[7-13]-BTN/SaV conjugate was effective in inhibiting cell growthand inducing apoptosis. These results show that specific delivery ofmultiple therapeutic agents to target cells enhances the effects of thetherapeutic agents and serves as an improved method for drug delivery.

EXAMPLE 1 Synthesis of MDML Conjugate

A MDML conjugate synthesis is described below. The MDML conjugatecomprises a plurality of tripartite molecules bound to a scaffoldmoiety. The tripartite molecules themselves comprise a targetingmolecule, a therapeutic agent and a scaffold binding moiety joinedtogether by plurality of linker molecules. Specifically, the synthesisof the PTX-BBN[7-13]-BTN/SaV conjugate is described, where thetripartite molecule comprises paclitaxel (PTX) as the therapeutic agent,a bombesin/gastrin-releasing peptide-(BNN/GRP-) receptor-recognizingpeptide (BBN[7-13] SEQ. ID NO. 1) as the targeting molecule and biotin(BTN) as the scaffold binding element and streptavidin (SaV) comprisesthe scaffold moiety. Two alternate approaches to the synthesis of thePTX-BBN[7-13]-BTN/SaV conjugate are illustrated in FIGS. 1A and 1B.

The synthesis of the tripartite molecules preferably incorporates alinker to join the targeting molecule, therapeutic agent and scaffoldbinding moiety to a central core, described below as lysine and diaminobenzoic acid. The synthesis described below uses polyethylene glycol(PEG) as the linker, although other linker molecules can be used. Thelinker molecules were used to reduce the effects of steric hindrance andto increase the overall yield of the synthetic reaction. It wasdetermined that a longer chain PEG molecule (3,400 molecular weight)worked well. The chemical and biological properties of polyethyleneglycols have been extensively studied and some useful characteristics ofthis polymer have been noted. These include aqueous as well as organicsolubilities, lack of immunogenicity, and favorable blood clearancepatterns and in vivo behavior. Furthermore, a heterobifunctionalderivative of the PEG was used in this synthesis, allowing the PEG toconnect the lysine and targeting molecule, therapeutic agent andscaffold binding moiety. The chemistry and properties of PEG aredescribed in J. M. Harris, Polyethylene Glycol Chemistry: Biotechnicaland Biomedical Applications, Plenum, New York, N.Y., 1992; and J. M.Harris and S. Zalipsky, Chemistry and Biological Applications ofPolyethylene Glycol, ACS Books, Washington, D.C., 1997 and in U.S. Pat.No. 5,643,575).

The synthesis of the PTX-BBN[7-13]-BTN/SaV conjugate using lysine as thecore molecule is illustrated in FIG. 1A. The procedure is initiated withthe synthesis of the 7-amino acid bombesin analog BBN[7-13] (SEQ ID NO.1). BBN[7-13], 1, was synthesized by SPPS and through a fluorenylmethoxycarbonyl (Fmoc) protocol (Synpep, Synpep, CA). Peptide 1 wascoupled to PEG to produce compound 2. Boc-lysine, 3, was condensed withPEG to produce compound 4. Compounds 2 and 4 were coupled together toproduce compound 5. The product 5 was treated with piperidine/DMF, andwas coupled to BTN-(sulfo)NHS to afford compound 6. Compound 6 wastreated with TFA yield compound 7, which was coupled to PEG to producecompound 8. After de-protection, compound 8 was mixed with PTX toproduce compound 9. Compound 9 was then mixed with a 25 mol % of SaV at4 degree Celsius and pH 5.0 with stirring for one hour. The finalproduct 10 was purified by RP-HPLC and identified by SE-HPLC on a G3000Pwxl column (TosoHaas, Montgomeryville, Pa.) pre-calibrated by molecularweight markers (Bio-Rad, Hercules, Calif.) (FIG. 2). As can be seen inFIG. 2C, the final product, compound 10, has a higher molecular weight(indicated by shorter retention times on the SE-HPLC column) than theun-reacted SaV (FIG. 2A) or compound 9 (FIG. 2B), indicating thesynthesis was successful.

The synthesis of the PTX-BBN[7-14]-BTN/SaV conjugate using diaminobenzoic acid as the core molecule is illustrated in FIG. 1B. Diaminobenzoic acid is a planar molecule with an central aromatic phenyl ringhaving a 1, 3, 5 substitution pattern, which may reduce steric hindranceamong the attached substituents. All coupling and deprotection stepsshown in this scheme are of standard procedures. All intermediate andfinal products are identified by standard analytical and spectroscopicmethods (NMR, mass spectroscopy, microanalysis, etc.). Solvents used areN,N-dimethyl formamide (DMF) and methylene chloride (DCM).Trifluoroacetic acid (TFA) and piperidine in DMF were used fordeprotection of the Boc and Fmoc groups, respectively. The procedure isinitiated with the synthesis of the 8-amino acid bombesin analogBBN[7-14] (SEQ ID NO. 2) 1 as described above. Peptide 1 is thenconnected to the central phenyl ring of diamino benzoic acid throughactive-ester chemistry using N,N-dicyclohexyl carbodiimide (DCC). TheN-Boc-N′-Fmoc-3,5-diamino benzoic acid used in this reaction has beenmade in our laboratory through standard protocols. The resultingcompound (3) undergoes two deprotection/coupling reactions to thecommercially available Fmoc-PEG-NHS and biotin, respectively, afford theprecleavage conjugate 5. The latter product is the cleaved from theresin, with concomitant removal of the Boc group by TFA to give 6. Afterpurification by reversed-phase high-performance liquid chromatography(RP-HPLC), compound 6 is coupled to a third molecule of the Fmoc-PEG-NHSlinker, which after removal of the Fmoc, is coupled to paclitaxel forafford the final product 7.

EXAMPLE 2 Cell Proliferation Assay

To evaluate the growth inhibitory effects of the PTX-BBN[7-13]-BTN/SaVconjugate, experiments were designed to compare the effect of theconjugate with the unconjugated paclitaxel, the BTN/SaV complex and amixture of free PTX and the BTN/SaV complex (FIG. 5). The low-receptordensity NCI-H1299 non-small cell lung cancer cells were used todemonstrate the extent of the sensitivity of a native, untransfectedcell line to treatment with the conjugate.

The HI299 cells were plated in 24 well plates at a density of 10,000cells/well and allowed to adhere for 24 hours. The cells were treatedwith 50 nM of the following: 1) no treatment (Untreated); 2)unconjugated paclitaxel (PTX); 3) biotin/streptavidin complex (BTN/SaV);4) a mixture of BTN/SaV and PTX; and 5) the PTX-BBN[7-13]-BTN/SaVconjugate (Conjugate) described in FIG. 1A. The drugs were removed fromthe cell monolayer after 3 hours and replaced with drug-free medium.After 96 hours, the cells were removed from the 24 well plates bytreatment with trypsin/EDTA and counted with a Coulter particle counter.All treatments were done in quadruplicate and the cell counts werenormalized to the untreated cells.

The PTX-BBN[7-13]-BTN/SaV conjugate demonstrated 37% growth inhibitioncompared to 0% growth inhibition by free PTX, and 1% and 4% inhibitionby BTN/SaV and BTN/SaV+PTX controls, respectively. The results indicatethat the PTX-BBN[7-13]-BTN/SaV was effective at inhibiting cell growthin the NIH HI299 cells where free PTX was not effective, suggesting theincreased targeting and increased drug delivery of thePTX-BBN[7-13]-BTN/SaV MDML conjugate provided enhanced cell inhibition.

EXAMPLE 3 Apoptosis Induction Assay

To evaluate the apoptosis-inducing ability of the PTX-BBN[7-13]-BTN/SaVconjugate, experiments were designed to compare the effect of theconjugate with the unconjugated paclitaxel, the BTN/SaV complex and amixture of free PTX and the BTN/SaV complex (FIG. 6). The low-receptordensity NCI-H1299 non-small cell lung cancer cells were used todemonstrate the extent of the sensitivity of a native, untransfectedcell line to treatment with the conjugate.

The HI299 cells were plated in 24 well plates at a density of 10,000cells/well and allowed to adhere for 24 hours. The cells were treatedwith 50 nM of the following: 1) no treatment (Untreated); 2)unconjugated paclitaxel (PTX); 3) biotin/streptavidin complex (BTN/SaV);4) a mixture of BTN/SaV and PTX; and 5) the PTX-BBN[7-13]-BTN/SaVconjugate (Conjugate) described in FIG. 1A. The drugs were removed fromthe cell monolayer after 3 hours and replaced with drug-free medium.After 96 hours, the cells were removed from the 24 well plates bytreatment with trypsin/EDTA and analyzed for Annexin V by flow cytometryusing an Annexin V-FITC kit (Biovision, Palo Alto, Calif.). Alltreatments were done in triplicate and presented as the percentage ofcells staining with Annexin V-FITC.

The PTX-BBN[7-13]-BTN/SaV conjugate showed a 7% induction of apoptosis,as compared to about 2.0%. 2.3%, 2.5% and 2.4% induction for untreatedcells, free PTX, the BTN/SaV complex and the BTN/SaV complex+PTXcontrols, respectively. The results indicate that thePTX-BBN[7-13]-BTN/SaV was effective at inducing apoptosis in the NIHHI299 cells where free PTX was not effective, suggesting the increasedtargeting and increased drug delivery of the PTX-BBN[7-13]-BTN/SaV MDMLconjugate provided enhanced cellular apoptosis.

Any patents or publications mentioned in this specification areindicative of the levels of those skilled in the art to which theinvention pertains. Further, these patents and publications areincorporated by reference herein to the same extent as if eachindividual publication was specifically and individually indicated to beincorporated by reference.

One skilled in the art will appreciate readily that the presentdisclosure is well adapted to carry out the objects and obtain the endsand advantages mentioned, as well as those objects, ends and advantagesinherent herein. The present examples, along with the methods,procedures, treatments, molecules, and specific compounds describedherein are presently representative of preferred embodiments, areexemplary, and are not intended as limitations on the scope of thedisclosure.

1. A method of treating an individual having a disease comprising thestep of administering to the individual in an amount sufficient to treatthe disease a compound comprising a plurality of tripartite moleculeslinked to a scaffold moiety, with each tripartite molecule comprising atargeting molecule for directing the compound to a defined population ofcells, a therapeutic agent and a scaffold binding element capable ofcomplementary interaction with the scaffold moiety, each of thetargeting molecule, the therapeutic agent and the scaffold bindingelement being joined by a linker molecule to a central core molecule,the targeting molecule and the therapeutic agent being selected based onthe disease to be treated.
 2. The method of claim 1 where each targetingmolecule of the compound is independently selected from the groupconsisting of a peptide, a receptor ligand peptide, abombesin/gastrin-releasing peptide receptor-recognizing peptide, asomatostatin receptor recognizing peptide, an epidermal growth factorreceptor recognizing peptide a monoclonal antibody or a portion thereof,a polyclonal antibody or a portion thereof, and a receptor recognizingcarbohydrate and the therapeutic agent is selected from the groupconsisting of paclitaxel and toxotere.
 3. The method of claim 1 wherethe disease is cancer.
 4. The method of claim 1 where the said compoundis administered by injection of intravenous infusion.
 5. The method ofclaim 2 where the receptor ligand peptide is selected from the groupconsisting of a bombesin/gastrin-releasing peptide receptor-recognizingpeptide, a somatostatin receptor recognizing peptide, and an epidermalgrowth factor receptor recognizing peptide.
 6. The method of claim 5where the receptor ligand peptide has a sequence selected from the groupconsisting of SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO.
 3. 7. The methodof claim 1 where each therapeutic agent is independently selected fromthe group consisting of a drug, an anti-tumor agent, a toxin, aradionuclide, and a metallic nuclei.
 8. The method of claim 1 where eachtherapeutic agent is independently selected from the group consisting ofpaclitaxel and toxotere.
 9. The method of claim 1 where the therapeuticmolecule is a diagnostic agent or a pharmaceutical agent.
 10. The methodof claim 1 where the scaffold binding element and the scaffold moietyform a binding pair and the binding pair is selected from the groupconsisting of biotin/avidin, biotin/streptavidin, hapten/antibody,ligand/receptor, enzyme/inhibitor and lectin/carbohydrate.
 11. Themethod of claim 1 where the scaffold binding element and the scaffoldmoiety are joined by a covalent bond.
 12. The method of claim 1 wherethe scaffold binding element and the scaffold moiety are joined by anon-covalent bond.
 13. The method of claim 12 where the scaffold bindingelement is biotin and the scaffold moiety is selected from the groupconsisting of avidin and streptavidin.
 14. The method of claim 1 wherethe linker molecule has at least one of the properties selected from thegroup consisting of solubility in organic solutions, solubility inaqueous solutions, lack of immunogenicity and long half life in thebloodstream.
 15. The method of claim 1 where the linker molecule is apolyethylene glycol molecule.
 16. The method of claim 15 where thepolyethylene glycol molecule is at least about 3400 molecular weight.17. The method of claim 15 where the polyethylene glycol moleculecontains at least two reactive groups.
 18. The method of claim 1 wherethe central core molecule is selected from the group consisting oflysine and diamino benzoic acid.
 19. The method of claim 1 where thetargeting molecule is the BBN[7-13] peptide, the therapeutic agent ispaclitaxel, the linker molecule is polyethylene glycol, the coremolecule is lysine, the scaffold binding element is biotin and thescaffold moiety is streptavidin.
 20. The method of claim 1 where thetargeting molecule is the BBN[7-13] peptide, the therapeutic agent ispaclitaxel, the linker molecule is polyethylene glycol, the coremolecule is diamino benzoic acid, the scaffold binding element is biotinand the scaffold moiety is streptavidin.